Elsevier

Biomedicine & Pharmacotherapy

Volume 94, October 2017, Pages 332-340
Biomedicine & Pharmacotherapy

Original article
Autophagy as a potential therapeutic target during epithelial to mesenchymal transition in renal cell carcinoma: An in vitro study

https://doi.org/10.1016/j.biopha.2017.07.070Get rights and content

Abstract

Cancer progression toward invasive and metastatic disease is aided by reactivation of epithelial–mesenchymal transition (EMT), involving transdifferentiation of epithelial cells into mesenchymal phenotype. This leads to increased migratory and stem cell-like features in the cells. These EMT cells are more resistant to chemotherapy and it is hypothesized that the phenomenon of autophagy induces resistance, providing a survival strategy for cells.

In the present study, we induced EMT-like phenotype in renal carcinoma cells and identified corresponding higher autophagy flux in these cells. The EMT transformed cells may be a representative of the resistant cancer stem cell(CSC)-like phenotype. Autophagy was identified as a potential mechanism of cell survival in these cells thus implying that autophagy inhibition can lead to enhanced cell death. We also observed that tumor cells especially EMT transformed cells, have been ‘primed’ to undergo autophagy by mTOR inhibition. We observed that combined use of autophagy inhibitor and temsirolimus (TEM) improved antitumor activity against RCC in EMT transformed metastatic cells. One of the approaches for inhibiting autophagy was the use of lysosomotropic anti-malarial drug, chloroquine (CQ) and we explored the therapeutic potential of combination of CQ and the mTOR inhibitor, TEM. EMT transformed cells showed increased cell cytotoxicity when autophagy was impaired by addition CQ with TEM. This led us to conclude that inhibition of autophagy with the current therapeutic regimen could be useful in targeting the EMT transformed cells along with the bulk tumor cells in RCC.

Introduction

Renal cell carcinoma (RCC) is the most common and aggressive renal neoplasia. RCC is potentially curable by surgical excision if diagnosed early; however, it becomes virtually incurable on metastasis to distant sites. Amongst the various subtypes of RCC, clear cell renal cell carcinoma (CCRCC) is the most prevalent form and constitutes 70–80% of all renal cancers [1].

Epithelial to mesenchymal transition (EMT) is a crucial process regulating the initial steps of metastatic progression. Therefore, key regulatory mechanisms and mediators of EMT may provide the new paradigms to develop novel therapeutics with broad clinical applicability. In RCC, it has been demonstrated that there is acquisition of EMT-like characteristics during the malignant transformation of renal epithelial cells [2], [3]. A recent study have considered and evaluated expression of EMT linked genes as a prognostic factor in the RCC [4]. EMT may lead to cancer cell invasion, metastasis, and drug resistance [5]. It has been reported earlier that anti-cancer therapies, including cytotoxic chemotherapy and pathway inhibitors can induce autophagy in most cancer cell lines [6]. Besides metabolic or cell intrinsic stresses, therapy-induced autophagy can limit the antitumor efficacy of a number of therapies [7]. Although autophagy can exert negative effects on tumor formation and progression, it has also been observed to support tumor survival and progression in many cases [8]. There exists a complex relation between autophagy, EMT and metastasis. Cells undergoing EMT require autophagy activation for survival during the metastatic spreading, on the other hand, autophagy acts as oncosuppressive signal to inhibit the early metastasis. During EMT, cancer cells go through morphological reprogramming and reorganisation of the cytoskeleton and promote autophagy for viability of potentially metastatic cancer cells [9], [10]. The activation of autophagic machinery has also been shown to cause reversion of the EMT phenotype [11], [12], [13], [14], [15]. Various evidences suggest that autophagy can control EMT through selective degradation of EMT proteins [16], [17]. In view of the dual role of autophagy in tumor progression, it is important to test its role in context dependent manner. It has been reported that the serine- threonine kinase, mTOR facilitates cell proliferation in presence of nutrients and growth factors while suppressing autophagy [18]. Pharmacologic inhibition of mTOR has been shown to supress tumor growth in both preclinical models and in patients with RCC. mTOR inhibitors (temsirolimus and evorilimus) have demonstrated a significant clinical activity in patients of advanced RCC [19], [20], [21] however the improvement in overall survival is not statistically significant [22], [23]. The duration of response is often limited and after sometime tumor cells become refractory to treatment and resistance develops in majority of patients [24]. Since mTOR inhibitors are known to induce autophagy therefore autophagy can be considered as a mechanism for tumor cells to escape therapy induced cell death and hence compromise efficacy of mTOR inhibitors in RCC.

In the present study, we identified autophagy as a potential mechanism of cell survival in metastatic RCC cells in vitro thus implying that autophagy inhibition can lead to enhanced cell death. We also observed that tumor cells especially EMT transformed cells, have been ‘primed’ to undergo autophagy by mTOR inhibition. It was our hypothesis that combined use of autophagy inhibitor and temsirolimus (TEM) would result in improved antitumor activity against RCC in EMT transformed metastatic cells. One of the approaches for inhibiting autophagy is the use of lysosomotropic anti-malarial drug, chloroquine (CQ) so we explore the therapeutic potential of combination of CQ and the mTOR inhibitor, TEM. EMT transformed cells showed increased cell death when autophagy was impaired by addition CQ with TEM. This led us to conclude that modulation of autophagy with the current therapeutic regimen could be useful in targeting the EMT transformed cells along with the bulk tumor cells in RCC.

Section snippets

Cell culture

In-vitro studies were done with A498 cell line procured from National Centre for Cell Sciences (NCCS), Pune India. The cells were further cultured in Dulbecco’s modified eagle’s medium-high glucose (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and with antibiotics (penicillin- streptomycin,1U/ml) at 37 °C at 5% CO2 in humidified incubator.

EMT induction in cell line

The A498cells were seeded in 6-well culture plates cells and treated with TGF- β (5 ng/ml) for 48 h. TGF-β treated cells were considered as EMT

EMT-like morphological change with TGF- β

A498 cells treated with TGF-β (TGF-β+ve) showed typical elongated, spindle shaped appearance characteristic of mesenchymal phenotype. The untreated cells (TGF-β-ve) retained the epithelial cobble stone appearance thus indicating induction of EMT in TGF-β+ve cells (Fig. 1).

Effect of mTOR inhibitor in RCC

mTOR inhibitor is the standard treatment regimen for RCC patients with metastases, but the response rate is variable. MTT assay showed that significant number of viable cells was found after treatment with 10 μM TEM (Fig. 2

Discussion

The mTOR inhibitors, temsirolimus and everolimus, are clinically approved for treatment of advanced RCC but their effect is not durable and majority of patients eventually experience disease progression while on treatment [24], [25], [26]. It is clear from the previous studies that proportion of EMT cells is higher in metastatic tumor and as evident from our result, mTOR inhibitors do not specifically target the EMT cells. The short-lived clinical response to mTOR inhibitors suggest that the

Acknowledgements

Authors acknowledge the technical staff of the Central sophisticated Instrumention Centre of PGIMER. Ms Mamta Singla received senior research fellowship from Indian Council of Medical Research (ICMR) Govt. of India.

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